Abstract
This study assessed the mechanical characteristics, biocompatibility and osteoconductive properties of an equine hydroxyapatite collagen (eHAC) bone block when applied as a bone substitute for lateral augmentation of rat mandible. 96 rats underwent lateral augmentation of the mandible, using two substitute bone blocks (eHAC or Bio-Oss ® spongiosa) or autologous bone grafts. Signs of inflammation, amount of bone formation and ingrowth of bone into the bone blocks were assessed at 1 and 3 months. eHAC blocks were mechanically rigid and could be fixed firmly and easily. Bio-Oss ® spongiosa blocks were brittle and fixation was difficult. eHAC and Bio-Oss ® spongiosa blocks were biocompatible and induced few or no signs of inflammation. Inflammation prevalence between the groups was not statistically different. Bone formation and bone growth into the blocks was significantly higher in eHAC than Bio-Oss ® spongiosa blocks, but lower than in autologous bone grafts (after 1 and 3 months). Regression analysis showed that the autologous bone graft predicted new bone formation at both time points. The eHAC block was only a predictor at 1 month; a trend was found at 3 months. The application of biodegradable membranes was not related to more bone ingrowth.
In maxillofacial implant surgery, bone augmentation procedures are frequently performed to obtain sufficient bone volume in the residual alveolar ridge to allow reliable placement of dental implants . For larger defects, autologous bone grafts harvested from the calvarium, iliac crest or mandible, either alone or in combination with alloplastic bone substitutes, are usually used. For small bony defects the augmentation procedure is often restricted to the use of either autologous bone or alloplastic bone substitutes as grafting material.
When autologous bone grafts are used, two major disadvantages have been reported in clinical studies. First, in the reconstruction of the severely resorbed alveolar crest by means of autologous bone grafts, there is a need for a second surgical site to harvest bone grafts. Harvesting an autologous bone graft can be associated with significant donor site morbidity, although in most cases complications and morbidity are low and of limited duration . Second, autologous bone grafts are subjected to resorption during healing. Several studies have shown up to 60% resorption after augmentation procedures of the alveolar ridge . The application of bone substitutes, such as deproteinized bovine bone (Bio-Oss ® ), may eliminate the problems of donor site morbidity and bone graft resorption . Bio-Oss ® has been applied as the sole augmentation material in procedures in small (one to two walled) bony defects of the alveolar ridge in many clinical cases. Bio-Oss ® can become incorporated into the recipient site and establish a close contact between newly formed bone and bone substitute material . A major disadvantage of several bone substitutes is that they are only available as granules or a paste. The mechanical characteristics of such bone substitutes do not allow the grafting material to be fixed stably to the alveolar ridge, but require a ‘gel’ or membrane to keep the material in place. These bone substitutes can be used for reconstructing a small bone defect (two or three wall defects), but are less useful when used for larger horizontal or vertical deficiencies. In an attempt to apply bone substitutes for larger reconstructions that lack a bone wall on three sites (onlay procedures for lateral or vertical augmentation), bone substitute blocks such as hydroxyapatite and tricalciumphosphate have been tested as onlay and inlay augmentation procedures in experimental cases. None of these products were evaluated for onlay mandibular augmentation. Bio-Oss ® collagen, which is available as a block, seems to lack the mechanical characteristics necessary for stable fixation when applied as a large onlay graft.
Recently, an equine hydroxyapatite collagen (eHAC) bone substitute (Geistlich Pharma AG, Wolhusen, Switserland) in block form has been developed. The reason for developing this new bone block was twofold: to make bone substitute in a block form with sufficient mechanical properties to enable stable fixation, which could be applied for lateral augmentation of the alveolar ridge; and to promote bone ingrowth through the presence of the collagen component. These promising characteristics, induced the authors to assess the mechanical characteristics, biocompatibility and osteoconductive capabilities of the eHAC blocks compared with Bio-Oss ® spongiosa bone blocks and autologous bone grafts when applied as a bone substitute for the lateral augmentation of a rat mandible.
Materials and methods
This study was approved by the Animal Studies Review Committee, and performed in accordance with the Institutional Guidelines (University Medical Center Groningen, Groningen, The Netherlands). Ninety-six rats (Sprague Dawley, male, aged 15–17 weeks, weight 350 g) were used in this study. The animals were housed in groups, and received standard laboratory food and drinking water.
Two types of substitute bone blocks were used, an experimental eHAC bone block and a Bio-Oss ® spongiosa block (control block). Both bocks were cut to the same dimensions (length × width × height: 4 mm × 4 mm × 3 mm). The eHAC bone block contains collagen, which is thought to make it less brittle and thus easier to fix to the alveolar crest. The collagen-containing block is thought to function as a scaffold that promotes ingrowth of bone into the blocks. Autologous bone grafts, measuring 3 mm × 2 mm × 1 mm were harvested from the mandibular angle area and transplanted to the mandibular area of the contralateral side. Harvesting of larger grafts mimicking the size of the experimental bone blocks was not possible owing to anatomical boundaries and the limited thickness of the mandibular angle. Autologous bone grafts were used as a second control for both substitute bone blocks, since they are considered to be the ‘gold standard’ for large augmentation procedures of the alveolar crest and because they are the solely material that is routinely used on a large scale.
Experimental design
The experimental design of this study has been well tested and accepted in previous studies . The animals were randomly separated into 4 groups (I, II, IIIA, IIIB) of 24 animals each ( Fig. 1 ). The split-mandible model design was used in group I animals. They underwent bilateral augmentation of the mandible with an eHAC bone block; one block was covered with a biodegradable membrane (BioGide ® ), the other was left uncovered. The split mandible model design was used in group II rats. They underwent bilateral augmentation of the mandible with a Bio-Oss ® spongiosa block; one block was covered with a biodegradable membrane (BioGide ® ), the other was left uncovered. Group IIIA rats underwent lateral augmentation of the mandible with an autologous bone graft taken from the contralateral side of the mandible; the bone graft was left uncovered. Group IIIB ratsunderwent lateral augmentation of the mandible with an autologous bone graft taken from the contralateral side of the mandible; the bone graft was covered with a biodegradable membrane (BioGide ® ).
Surgical procedure
All surgical procedures were performed by one investigator, helped by two assistants, and timed. All animals were anaesthetized with nitrous oxygen–isoflurane. After shaving and disinfecting the mandibular and cervical areas, a submandibular incision was made to expose the masseter muscle. The muscle was incised along the submandibular border and the masseter muscle including the periosteum, was raised. Care was taken not to injure the facial nerve, parotid duct. No periosteal release was necessary to create a pocket large enough to accommodate the substitute bone blocks. In group I, an eHAC bone block was fixed on the lateral side of the mandible with a 6.0 mm long 1.0 mm diameter titanium microscrew (KLS Martin Group, Huizen The Netherlands). The graft was covered with a biodegradable membrane (Bio-Gide ® ) in such a way that at least 2 mm of the mandibular bone adjacent to the grafted area was covered ( Fig. 2 ). The wound was closed with 4-0 resorbable sutures in two layers. On the contralateral side of the mandible, a second eHAC block was positioned and fixed; no biodegradable membrane was used. The same procedure was performed to fix two Bio-Oss ® spongiosa blocks, to both lateral sides of the mandible in group II. In the autologous bone graft groups, the graft was harvested from the angle of the mandible in an identical surgical procedure, then transplanted to the contralateral side of the mandible, and fixed with a microscrew. Similar to the substitute bone block groups, the autologous bone grafts were covered with a biodegradable membrane in rats belonging to group IIIB and left uncovered in group IIIA. For postoperative pain relief, a single dose of carprofen (4.0 mg/kg) was given. The animals were housed in groups of six and were given a soft diet for 3 days postoperatively after which they returned to standard food pellets. Water was given ad libitum .
After 1 and 3 months, 12 animals in each group were anesthetized by nitrous oxygen–isoflurane inhalation and killed by a lethal intracardial injection of pentobarbital. The mandibles were explanted and the excess soft tissue carefully removed. The samples were fixed in a 4% phosphate buffered formalin solution to await analysis.
Histology and histomorphometry
The samples were dehydrated in series of ethanol and embedded through resin infiltration of methylmethacrylate. The central point of each sample (the head of the miniscrew) was identified and the samples were cut in half. One half was used for histological analysis and histomorphometric measurements. The samples used for histological and histomorphometric analysis were cut into sections of 300 μm, mounted on opaque acrylic slides and ground to a final thickness of 50 μm. They were stained with Azur II and Pararosaniline. About six slices were made from each sample. The central most section was used for both histological analysis and histomorphometric measurements.
Histological analysis was performed on all samples to obtain a qualitative impression of the amount of bone formation and/or resorption in and around the bovine bone blocks and the biocompatibility of the biomaterial used. The amount of bone formation and signs of inflammation were scored as: none (0), little (1+), moderate (2+) and much (3+). Inflammation was evaluated by quantifying the number of lymphocytes, granulocytes, plasma cells and macrophages present and scored accordingly: none (0–5%), little (5–30%), moderate (30–60%), much (>60%). The location from where bone formation began as well as possible bone ingrowth into the biomaterial was noted. In the autologous bone graft groups, the osteoblastic and osteoclastic activity was noted as a sign of bone remodelling. Bone formation, bone ingrowth and signs of inflammation were studied in relation to the presence or absence of a biodegradable membrane. To obtain an objective measurement of bone formation, all histological samples underwent histomorphometric analysis.
Histomorphometric analysis was performed to quantify the amount of bone formation and bone ingrowth into the substitute bone blocks and to compare these results with the amount of bone formation after lateral augmentation of the rat mandible using autologous bone grafts. First, a slice of a particular sample was placed on a light box, where after images were recorded and stored with a digital camera (12.5× magnification) mounted on a light microscope. An image processing program (Image-J ® , National Institute of Health, Bethesda, Maryland, USA) was used to digitally colour the different tissues of interest (pre-existing bone, new bone, Bio-Oss ® , connective tissue). The areas of these coloured surfaces were digitally measured and expressed as surface per mm 2 ( Fig. 3 ). Newly formed bone was defined as new bone formation noted on the surface of the rat mandible, at the interface of pre-existing bone and bone substitute, and bone formation into the bone substitute.
Statistical analysis
Differences between the eHAC block, spongiosa bone blocks and autologous bone grafts in operating time and mean percentage new bone formation at 1 and 3 months were examined using one-way Analysis of Variance (ANOVA), with post-hoc Bonferroni testing. The assumption of normality was mildly violated, but sample sizes within groups were equal and large enough to perform ANOVA adequately.
Differences between the groups in prevalence of bone formation and inflammation were examined with the χ 2 test. Dichotomization of no and little bone formation versus moderate to much bone formation was used and dichotomization of no inflammation versus little–moderate–much inflammation. Differential dichotomization was applied to create most equal groups and enhance statistical power. When significant differences were found between the three groups, additional χ 2 analyses with Bonferroni correction for alpha (i.e. alpha divided by the number of tests; alpha = 0.05/3 = 0.017) were performed to investigate the nature of the differences.
Linear regression analysis was performed to investigate the impact of treatment on percentage new bone formation at 1 and 3 months. For this purpose, two dummy variables were created with the control block as reference group. These dummies together with the potential confounders, operation time and inflammation, were entered in the model.
Differences in mean percentage new bone formation within the eHAC block, spongiosa bone block and autologous bone grafts stratified by use of membrane were examined with independent-samples t -test.
A p -value <0.05 was used for all tests to indicate statistical significance. All statistical analyses were performed using SPSS version 17.0.
Results
All test animals recovered uneventfully after the surgical procedure and no animals needed to be excluded from the study due to postoperative complications.
No significant differences were noted in the length of the surgical procedures between rats from the three groups offered at 1 month ( F (2,69) = 1.26, p = .29) and at 3 months ( F (2,69) = 0.28, p = .76). The overall average operating time was 26.3 min for rats offered at 1 month and also 26.3 min for rats offered at 3 months. Average operation times in minutes for rats offered at 1 month and at 3 months for each of the groups were as follows: eHAC 26.8 (range 21–34) and 25.8 (range 22–34), Bio-Oss ® spongiosa 26.7 (range 22–35) and 26.5 (range 22–34), autologous bone graft 25.4 (range 19–34) and 26.5 (range 22–34).
Handling characteristics of blocks
In groups I and II, 96 substitute bone blocks were fixed to the rat mandible, 48 eHAC blocks and 48 Bio-Oss ® spongiosa blocks. All blocks were fixed with a screw inserted by a screwdriver until the surgeon felt that the block was firmly fixed to the mandible. The eHAC blocks were rigid and could not be crushed by pinching forces between two fingers. All blocks could be firmly fixed to the rat mandible and no block had to be replaced due to breakage during fixation. By contrast, the Bio-Oss ® spongiosa blocks were brittle and could be easily crushed by pinching forces between two fingers. When fixed to the mandible the miniscrew could easily be driven through the blocks and the blocks broke when too large a pressure was applied. Seventeen of 48 (35%) Bio-Oss ® spongiosa blocks had to be replaced during the surgical procedure due to breakage.
Histological analysis
One month after augmentation, the sites grafted with an eHAC block showed no to little signs of inflammation in 96% of cases (23/24 samples). In 4% of cases (1/24 samples) microscopic analysis showed more than 60% inflammatory cells (3+). After 3 months, 16/24 samples (67%) showed no signs of inflammation, while 1 sample (4%) showed much inflammation. New bone formation was seen in all samples after 1 and 3 months. All samples showed bone formation starting at the interface of pre-existent bone and bone substitute. Ingrowth of bone into the bone block was observed in 63% of cases (15/24 samples) after 1 month and in 67% of cases (16/24 samples) after 3 months ( Fig. 4 a). In almost all cases, ingrowth of bone into the bone blocks was observed, starting from the surface of the pre-existing bone where the newly formed bone bridges the gap between pre-existing bone and bone substitute and then growths into the cavities of the bone substitute. The use of a biodegradable membrane did not result in a higher amount of bone formation or more bone ingrowth into the bone block nor did it result in less inflammation ( Table 1 ).
| Number of animals | Number of surgical sites (split-mouth model) | Bone formation | Location of bone formation | Bone ingrowth | Signs of inflammation | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| M+ (12) | M− (12) | M+ | M− | M+ (12) | M− (12) | |||||||||
| 1 month | 12 | 24 | 0+ | 0 | 0 | 0 | At the Interface of pre-existent bone and biomaterial | 15/24 | 9 | 6 | 0+ | 12 | 5 | 7 |
| 1+ | 9 | 5 | 4 | 1+ | 11 | 6 | 5 | |||||||
| 2+ | 14 | 7 | 7 | 2+ | 0 | 0 | 0 | |||||||
| 3+ | 1 | 0 | 1 | 3+ | 1 | 1 | 0 | |||||||
| 3 months | 12 | 24 | 0+ | 0 | 0 | 0 | At the Interface of pre-existent bone and biomaterial | 16/24 | 10 | 6 | 0+ | 16 | 7 | 9 |
| 1+ | 11 | 5 | 6 | 1+ | 5 | 3 | 2 | |||||||
| 2+ | 11 | 5 | 5 | 2+ | 2 | 1 | 1 | |||||||
| 3+ | 2 | 2 | 1 | 3+ | 1 | 1 | 0 | |||||||
Bone formation in the sites grafted with a Bio-Oss ® spongiosa block was less than in sites grafted with an eHAC block ( p < 0.05). No bone ingrowth was seen after 1 month (0/24) and after 3 months it was observed in only 13% of cases (3/24). New bone formation was mainly seen at the interface of pre-existing bone and bone substitute. No significant differences were noted between grafts covered with a biodegradable membrane and grafts placed directly under the periosteum-masseter muscle flap at 1 and 3 months ( p < 0.05) ( Table 2 , Fig. 4 b).
| Number of animals | Number of surgical sites (split-mouth model) | Bone formation | Location of bone formation | Bone ingrowth | Signs of inflammation | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| M+ (12) | M− (12) | M+ | M− | M+ (12) | M− (12) | |||||||||
| 1 month | 12 | 24 | 0+ | 0 | 0 | 0 | At the Interface of pre-existent bone and biomaterial | 0/24 | 0 | 0 | 0+ | 11 | 4 | 7 |
| 1+ | 20 | 9 | 11 | 1+ | 8 | 5 | 3 | |||||||
| 2+ | 4 | 3 | 1 | 2+ | 2 | 1 | 1 | |||||||
| 3+ | 0 | 0 | 0 | 3+ | 3 | 2 | 1 | |||||||
| 3 months | 12 | 24 | 0+ | 0 | 0 | 0 | At the Interface of pre-existent bone and biomaterial | 3/24 | 0 | 3 | 0+ | 13 | 5 | 8 |
| 1+ | 11 | 5 | 6 | 1+ | 10 | 6 | 4 | |||||||
| 2+ | 11 | 5 | 6 | 2+ | 0 | 0 | 0 | |||||||
| 3+ | 2 | 2 | 0 | 3+ | 1 | 1 | 0 | |||||||
Histological analysis of the sites grafted with autologous bone grafts showed similar amounts of bone formation at 1 and 3 months ( Table 3 , Fig. 4 c). Bone formation was found mainly at the interface of pre-existent bone and bone graft and around the miniscrew. Osteoclastic and osteoblastic activity (bone remodelling) was mainly seen at 1 month (11/24) after grafting. After 3 months, bone remodelling activity decreased to 5/24 samples. Signs of inflammation were non-existent (0+) or mild (1+), and were comparable at 1 and 3 months. The amount of inflammation of autologous bone grafts was comparable with both types of substitute bone blocks. There were no significant differences between grafts covered with a biodegradable membrane and grafts left uncovered.
| Number of animals | Number of surgical sites (unilateral surgical site) | Bone formation | Location of bone formation | Bone ingrowth | Signs of inflammation | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| M+ (12) | M− (12) | M+ | M− | M+ (12) | M− (12) | |||||||||
| 1 month | 24 | 24 | 0+ | 0 | 0 | 0 | At the Interface of pre-existent bone and biomaterial | 11/24 | 6 | 5 | 0+ | 17 | 8 | 9 |
| 1+ | 14 | 9 | 5 | 1+ | 7 | 4 | 3 | |||||||
| 2+ | 9 | 2 | 7 | 2+ | 0 | 0 | 0 | |||||||
| 3+ | 1 | 1 | 0 | 3+ | 0 | 0 | 0 | |||||||
| 3 months | 24 | 24 | 0+ | 0 | 0 | 0 | At the Interface of pre-existent bone and biomaterial | 5/24 | 3 | 2 | 0+ | 18 | 9 | 9 |
| 1+ | 20 | 11 | 9 | 1+ | 6 | 3 | 3 | |||||||
| 2+ | 4 | 1 | 3 | 2+ | 0 | 0 | 0 | |||||||
| 3+ | 0 | 0 | 0 | 3+ | 0 | 0 | 0 | |||||||
There were differences between the three groups in new bone formation at 1 month ( χ 2 (2, n = 72) = 10.51, p = 0.05), with the prevalence of moderate to much new bone formation being significantly higher in the e-HAC block compared with the Bio-Oss block (63% versus 17%, χ 2 (1, n = 48) = 10.54, p = .001). The prevalence did not differ statistically between the e-HAC block and the autologous block (63% versus 42%, χ 2 (1, n = 48) = 2.09, p = .15) and between the Bio-Oss and autologous block (17% versus 42%, χ 2 (1, n = 48) = 3.63, p = .057). Significant differences between the 3 groups also emerged at 3 months ( χ 2 (2, n = 72) = 9.26, p = .010), but the nature of the differences was different. The e-HAC block and Bio-Oss block did not differ in prevalence of moderate to much new bone formation (54% versus 54%, χ 2 (1, n = 48) = 0.0, p = 1.0). In contrast, the prevalence within the e-HAC block was significantly higher compared with the autologous block (54% versus 17%, χ 2 (1, n = 48) = 7.38, p = .007). And similar results were found for the Bio-Oss block compared with the autologous block (54% versus 17%, χ 2 (1, n = 48) = 7.38, p = .007).
The prevalence of inflammation at 1 month was lower in the autologous bone block (29%) compared with the e-HAC block (50%) and control block (54%), but this difference was not statistically different ( χ 2 (2, n = 72) = 3.49, p = 0.18). Similar results were found at 3 months, when the autologous block (25%) displayed the lowest prevalence compared with the eHAC block (33%) and control block (46%), which was not significant ( χ 2 (2, n = 72) = 2.33, p = .31). No additional χ 2 analyses were performed.
Histomorphometric analysis
The mean percentage new bone formation is depicted in Fig. 5 and Table 4 . The mean percentage new bone formation at 1 month was significantly different across the three groups ( F (2,69) = 49.59, p < .001). The highest formation of new bone was found in the autologous bone group (19%), followed by the e-HAC block (6%), and finally the Bio-Oss ® block (4%) (all group comparisons significant with p < .005). ANOVA was also significant at 3 months ( F (2,69) = 82.89, p < .001), but at this time point the e-HAC block (5%) did not differ from the Bio-Oss block (1%) in mean percentage new bone formation ( p = .13). The group with the autologous block (12%) displayed a significantly higher mean percentage of bone formation compared with the other two groups (both p < .001).
